Michael's Research

• Mycobacterium Tuberculosis H37Rv

sbPSTP (2CM1) Research:

• Week 12:

  • FPLC Ahh! - Embed the image instead of linking to it....... Dr. B

    • 

      20111117_MichaleEaston_FPLCresult_PSTP.bmp
      

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  • Protein Characterization

    • 

• Week 11:

  • Protein Purification

    • 

• Week 10:

  • Protein Expression

    • Lysed the pellets with lysis reagent and spun down the contents

  • PCR: Primer Design

    • Secondary PCR for 2nd and 3rd attempt, running Gel Sunday

• Week 9:

  • Protein Expression

    • Pellets are highlighted

    • Pellet weight: .97 +1.59 = 2.56g
    • 
    • At OD600
      • 1st measure 12:00 0.0628265235129
      • 2nd measure 12:10 0.146219267443
      • 3rd measure 2:20 0.391527989213

• Week 8:

  • PCR: Primer Overlap

    • Made new Primary PCR after repeated failures of last week

  • Virtual Screening

• Week 7:

  • PCR: Primer Overlap

    • Lane 1: 100bp Ladder; Lane 2: Secondary PCR

    • Primer overlap for sbPSTP, comparing to a 100 bp ladder. 2nd run of secondary PCR failed.

• Week 6:

  
  

  PCR: Primer Overlap

  • Lane 1: 100bp Ladder, Lane 2: Primary PCR; Lane 3: Secondary PCR

  • Primer overlap for sbPSTP, comparing to a 100 bp ladder. The primary PCR looks succesful as there should be a smear; however the secondary PCR has extra bands, where it should only have a band at ~700bp

• Week 5:

  • PCR Primer Design for Primer Overlap Assembly PCR

    • Protein: sbPSTP
    • Organism: Mycobacterium Tuberculosis PSTP

    • --------------------------------------------------------------------------------
      |             Helix Systems -- Center for Information Technology               |
      |                            http://helix.nih.gov                              |
      |   National Institutes of Health, Department of Health and Human Services     |
      |                                                                              |
      |             DNAWorks Web Site: http://helixweb.nih.gov/dnaworks              |
      --------------------------------------------------------------------------------
      |                                                                              |
      |              Send all correspondence to webtools@helix.nih.gov               |
      --------------------------------------------------------------------------------
       Job started on 10/02/2011 at 19:39:44
       Job name: ME100211sbPSTP                                                       
       Output will be sent to maeaston25@gmail.com          
                          Starting   1 trial...
       
       ----------------------------------------------------------------
                          PARAMETERS FOR TRIAL 1                             
       ----------------------------------------------------------------
                     Total Size Of Gene ......... 723 nt                        
                     Protein Residues ........... 241                           
                     Mutatable Residues ......... 235                           
                     Fixed Nucleotides .......... 18 nt                         
                     Oligo Size ................. 60 nt                         
                     Annealing Temp ............. 62 +/- 1*C                    
                     Oligo Concentration ........ 1.00E-7 M                     
                     Sodium Concentration ....... 5.00E-2 M                     
                     Mg2+ Concentration ......... 2.00E-3 M                     
                     Codon Frequency Threshold .. 10%                           
                     Repeat Threshold ........... 8 nt                          
                     Mispriming Threshold ....... 8/18 (6 exact) nt             
              100 optimization rounds, best =     3.436 Rep =   6 Mis =   0
       
       The total codon usage score ...........     0.000
       The total length score ................     0.000
       The total melting temperature score ...     0.000
       The total repeat score ................     0.000
       The total pattern score ...............     0.000
       The total mispriming score ............     0.000
       The total AT content score ............     0.000
       The total GC content score ............     0.000
       The total fixed gap score .............      N/A
                      The OVERALL score ......     0.000
       
      Finished trial   1 : rate =   0.2 min/cyc : time remaining =   0.0 min.
       
                                FINAL SUMMARY FOR   1 SOLUTION   
      --------------------------------------------------------------------------------
        #    Tm   Len  |    Score   TmRange  Short    Long   #Olig  #Repeat #Misprime
       
        1    62    60  |    0.000       1.8      15      60      18       0       0
       
      --------------------------------------------------------------------------------
      |             Helix Systems -- Center for Information Technology               |
      |                            http://helix.nih.gov                              |
      |   National Institutes of Health, Department of Health and Human Services     |
      |                                                                              |
      |             DNAWorks Web Site: http://helixweb.nih.gov/dnaworks              |
      --------------------------------------------------------------------------------
       
      Finished </span>

First Protocols:

• Week 4:

• Practice PCR: PNIC-bsa4

  • Lane 1: 100bp Ladder; Lane 2: 100bp Ladder; Lane 3: 1:10 Dilution; Lane 4: 1:100 Dilution; Lane 5: 1:1000 Dilution; Lane 6: Control

  • No Yield, possible contamination of lane 3

  • Lane 1: Empty; Lane 2: 100bp Ladder; Lane 3: 1:100 Dilution; Lane 4: 1:1000 Dilution; Lane 5: 1:10000 Dilution; Lane 6: Control

  • No Yield

• Virtual Screening Refresher:

  • 2nd Run submitted, still waiting to be run

• Week 3:

• REDigest

  • Lane 1: empty; Lane 2: 1kb Ladder; Lane 3: uncut plasmid; Lane 4: EcoRI; Lane 5: PvuII; Lane 6: EcoRI & PvuII

  • PvuII cut the plasmid pGBR22 properly in both lanes 5 and 6. EcoRI failed to cut in either lane 4 or 6 and causing multimer bands to form in lane 4.

• Week 2:

• Practice protocol for PCR

  • 1st attempt of pcr with no results

  • Lane 1: Empty; Lane 2: 100bp Ladder; Lane 3: 1ul 1:1000; Lane 4: 10ul 1:1000; Lane 5: 10ul 1:100; Lane 7: overflow

  • 2nd run of PCR with pGBR22 template showing ~1000bp. Output is stronger in lanes 4&5 where the intial concentration of template is higher. lane 6 yielded nothing, being the control. Lane 7 is possible over flow from the other lanes.

• Week 1:

  • DNA Sequencing

1. pGBR22 M13F
NNNNNNNNNGTATACGACTCACTATAGGGCGAATTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCCGCGGGATTTTAGTGATGGTGATGGTGATGACCGAGCAAAGAGTGGCGTGCAA
TGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACATCCAGTTTGCGGTCAACATAGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTG
AATTCACACAAATAGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCAGTGTTGGGTTCCCAGCCCTGTGTCTTCTTCTG
CATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATTGTAGATGAAACAGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGA
TCCTCTCCCATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTCAGGGTACTTGGTGAATGGTATGCTTCCGTATTGAGACAGTGGTGATAAAATATCCCAAGCAAATG
GCAGAGGTCCACCCTTGGTGACAGTGAGCTTTACCGTCTGCTCCCCCTCGTAAGGCTTTCCTTTTCCATCGCCTTCGACCTCAAAGTAGTGTCCATTGACCGTGCCTGACATATAAACCTTG
TAGGTCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTAGTGCGGCCGCCTGCAGGTCGACCATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTTG
AGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGC
CTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCNGCTTTCCAGTNGGAAACCTGTCNTGCCAGCTGCATNANGAATCGNCANNNNNNGGGNNANGNNGNTT
GCGTNTNGGNNCTNNTNNNCTNCNTNNNNNNNTGACTCGCTGNNNNNGNCNNNGNTGNNNNNNNNNNNNCANNTNNNTNNNNGNNNNNCGNNNNNNNNNNNCNGGNNNNNCNGAAANANNNN
NNNNNNNNANNNNNNNNNNNNNNNNCNNNAANNNNNNNNNNNTNGNNTTTNNNNNNNNN
NCNCCCCCNNNNNN

2.pGBR22 M13R
NNNNNNNNNNTGNNNATAGAATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTTTGATTGATTGAAGGAGAAATATCATGA
GTGTGATCGCTAAACAAATGACCTACAAGGTTTATATGTCAGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAAGCTC
ACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATT
CCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGGTGTGA
ACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAA
GGAGGTGGTTACTATTTGTGTGAATTCAAATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTACAC
ATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCACTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCC
TATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGNGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAG
CGAANAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGNNAATGGNCGCNNCCTGTAGCGNNGCATNNCGCGGNNGGGNGNNGNNGNNCNNNCANCGNGACNCTACNNN
NNNNNNCCNNNNNCCGNNNCTTTCGNTTNNNTNCNNTTNNNNGCCNCNTNNNNNTTNCCCNNNANNTNNAANNGGGNNNCNTTNGNNNNCNANTNNNNNTNNGNNNNNNACNNNNAANCNNN
NNNNNGNNNNNNNNNNNNNGNNNNNNNCCCNNNNNNANGNNNTNNCCNNNNNNNCNNTNNNNNNN

The more you learn:

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